Constitutive activity of JNK2 alpha2 is dependent on a unique mechanism of MAPK activation.
c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2alpha2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2alpha2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2alpha2 autophosphorylation and autoactivation. We find that JNK2alpha2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the alpha-region. Alanine scanning mutagenesis of the alpha-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2alpha2 dimerization rendering JNK2alpha2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2alpha2 homodimerization. We also show that a kinase-inactive JNK2alpha2 mutant can interact with and inhibit wild type JNK2alpha2 autophosphorylation, suggesting that JNK2alpha2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2alpha2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2alpha2 activity and that prevention of dimerization may decrease JNK2alpha2 induced tumorigenic phenotypes.
Nitta
Ryan T
RT
Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305, USA.
Chu
Albert H
AH
Wong
Albert J
AJ
D000409:Alanine / Q000737:chemistry; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D020935:MAP Kinase Signaling System; D048056:Mitogen-Activated Protein Kinase 9 / Q000378:metabolism*; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism
D003432:Cross-Linking Reagents; D048056:Mitogen-Activated Protein Kinase 9; D000409:Alanine